Ultrathin‐FeOOH‐Coated MnO2 Sonosensitizers with Boosted Reactive Oxygen Species Yield and Remodeled Tumor Microenvironment for Efficient Cancer Therapy

Abstract Sonodynamic therapy (SDT) typically suffers from compromised anticancer efficacy owing to the low reactive oxygen species (ROS) yield and complicated tumor microenvironment (TME) which can consume ROS and support the occurrence and development of tumors. Herein, ultrathin‐FeOOH‐coated MnO2 nanospheres (denoted as MO@FHO) as sonosensitizers which can not only facilitate ultrasound (US)‐triggered ROS but also tune the TME by hypoxia alleviation, H2O2 consumption as well as glutathione (GSH) depletion are designed. The FeOOH coating will boost the production yield of singlet oxygen (1O2) and hydroxyl radicals (•OH) by inhibiting the recombination of US‐initiated electron–hole pairs and Fenton‐like reaction, respectively. Additionally, the catalase‐like and GSH peroxidase‐like activities of MO@FHO nanospheres enable them to break the TME equilibrium via hypoxia alleviation and GSH depletion. The combination of high ROS yield and fundamental destruction of TME equilibrium results in satisfactory antitumor outcomes, as demonstrated by the high tumor suppression efficacy of MO@FHO on MDA‐MB‐231‐tumor‐bearing mice. No obvious toxicity is detected to normal tissues at therapeutic doses in vivo. The capability to modulate the ROS production and TME simultaneously can afford new probability for the development of advanced sonosensitizers for synergistic comprehensive cancer therapy.

by DCT-700, SZWELLD. Photoluminescence decay profiles was detected by FLS980, EDINBURGH INSTRUMENTS. Zeta potential was determined on Brookhaven Zetasizer Nanoseries (EliteSizer) using deionized water as medium. Particle sizes of MO-PD and MO@FHO-PD were measured on dynamic light scattering (DLS, NanoBrook 90Plus PALS) using undiluted Research Grade EU Approved Serum as medium.

ROS generation under ultrasound irradiation:
Objective to 1 O 2 detection, 3 mL of MO@FHO solution (0.05 mg mL −1 in deionized water) was mixed with 10 μL of 1,3diphenylisobenzofuran (DPBF, 10 mM in DMSO). Then the mixture was exposed to US irradiation at fixed time intervals (1 min) until 10 min. The concentration of DPBF at different time points was recorded by UV-vis spectra. For •OH detection, 3 mL of MO@FHO solution (0.05 mg mL −1 ) was mixed with 10 μL of o-phenylenediamine (OPD, 0.1 M in deionized water). Then the mixture was exposed to US irradiation at fixed time intervals (2 min) until 10 min. UV-vis spectra were measured to monitor the ascending trend at different time points.
And ESR technology was also employed to detect the generated ROS. In this case, 100 μL of MO@FHO solution (0.1 mg mL −1 ) was mixed with 20 μL of 2,2,6,6-tetramethylpiperidine (TEMP, for 1 O 2 detection) or 30 μL of 5,5-dimethyl-pyrroline-N-oxide (DMPO, for ·OH detection). The mixture was exposed to US irradiation for 5 min and immediately detect characteristic peak signals by ESR technology. In addition, the reactive oxygen species (ROS) generating ability of control (without sonosensitizers) and MO groups was detected by the same way. All experiments were carried out under fixed US parameters (1 MHz, 1.5 W cm −2 ).
Fenton-like activity: 3 mL of MO@FHO solution (0.05 mg mL −1 in deionized water) was incubated with H 2 O 2 (10 −4 M), and 10 μL OPD (0.1 M in deionized water) was added as an indicator of •OH. Then the mixture was exposed to US irradiation (1 MHz, 1.5 W cm −2 ) at fixed time intervals (2 min) until 10 min. UV-vis spectra of the samples at different time points were scanned to monitor the ascending trend. The Fenton-like activity measurement of control and MO groups was detected by the same way.
GSH peroxidase-like activity: 3 mL of MO@FHO solution (0.05 mg mL −1 in dimethyl sulfoxide) was incubated with glutathione (GSH, 10 −4 M) for 1 h, and then 100 μL 5,5ʹdithiobis (2-nitrobenzoic acid) (DTNB, 3 mg mL −1 in dimethyl sulfoxide) was added as an indicator of GSH. After standing for 1min, UV-vis spectra of the sample were scanned to monitor the GSH depletion. The GSH peroxidase-like activity measurement of control and MO groups was detected by the same way.

Catalase-like activity:
The H 2 O 2 (5×10 −4 M) was added into the phosphate buffered saline (PBS, pH=6.5) solution of MO@FHO (0.1 mg mL −1 ). Then, dissolved O 2 content increment in real-time was monitored by a portable dissolved oxygen meter (JPSJ-605F, INESA). The catalase-like activity measurement of control and MO groups was detected by the same way.
Electrochemical measurements: Amperometric current-time (I-t) and linear sweep voltammetry (LSV) curves were collected by using an electrochemical workstation (CHI 760).
All the electrochemical measurements were characterized in a three-electrode system, in which PBS with/without H 2 O 2 addition (10 −4 M) as the electrolyte, saturated calomel as the reference electrode and graphite rod as the counter electrode. On day 0 of the experiment, the nude mice were anesthetized by inhaling 2% animal isoflurane. After inhalation anesthesia, ultrasound images were used to calculate the tumor volumes of each nude mice by the following equation: Then groups 3 and 4 were injected with 12.5 mg kg −1 of MO-PD, groups 4 and 6 were injected with 12.5 mg kg −1 of MO@FHO-PD, and groups 1 and 2 were injected with equal volume of normal saline. After i.v. injection, the tumors of groups 2, 4 and 6 were treated with US irradiation (1 MHz, 1.5 W cm −2 , 2 min) immediately. The procedures were repeated on day 3, 7, 10, 17, and 21 respectively, and the tumor volumes on day 0, 7, 14, and 21 were recorded.
On day 21 of the experiment, all nude mice were euthanized with excessive anesthesia and dissected. The heart, lung, liver, spleen and kidney of each group were collected for Hematoxylin-eosin (H&E) observation. And tumor tissues were collected to execute H&E, vascular endothelial growth factor (VEGF), Ki67 and hypoxia-inducible factor (HIF-1α) immunohistochemistry staining.
Statistical Analysis: All experiments were performed at least three times, while the results are presented as mean ± SD. The significant difference between different groups was calculated by Two-way ANOVA with Bonferroni posttests. Significance is indicated by P < 0.05 (*P < 0.05, **P < 0.01, ***P <0.001). All data were analyzed by GraphPad Prism and Excel.